Targeting a viral macrodomain: Design, Structure-Based Optimization and Antiviral Evaluation of Nanomolar Inhibitors for Mac1 of SARS-CoV-2

Targeting a viral macrodomain: Design, Structure-Based Optimization and Antiviral Evaluation of Nanomolar Inhibitors for Mac1 of SARS-CoV-2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting a viral macrodomain: Design, Structure-Based Optimization and Antiviral Evaluation of Nanomolar Inhibitors for Mac1 of SARS-CoV-2 Ralf Fliegert, Maximilian Sandmann, Sahra Tajdar, Simon Sander, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7063434/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Enzymatically active viral macrodomains of (+)ss-RNA viruses mediate immune evasion by countering ADP-ribosylation and are therefore promising druggable targets. We tested ADP / ADP-ribose analogs for inhibition of Mac1 of SARS-CoV-2 and determined affinity and binding mode of active compunds. The SAR showed, that replacement of either the distal ribose of ADPR by a small alkyl group or the adenine N7 by carbon improved inhibitory potency, resulting in the development of β-methyl-GS-441524-diphosphate, a nanomolar inhibitor that exhibits high selectivity (>1000-fold) over human MacroD1 and MacroD2. Addition of C 11 -acyloxybenzyl (AB)-masking groups yielded a membrane permeable, lipophilic prodrug that inhibits SARS-CoV-2 in cell culture (EC 50 0.06 μM) while exhibiting low cytotoxicity (CC 50 > 50 μM). Replacement of the terminal methyl phosphate with an ethyl phosphonate increased stability of the prodrug with little effect on toxicity and antiviral potency (EC 50 = 0.03 μM), making it the first membrane-permeable nucleotide-based prodrug against viral macrodomains. Biological sciences/Biochemistry/Enzymes Biological sciences/Drug discovery Biological sciences/Microbiology/Virology/SARS-CoV-2 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation20250722.pdf Supplementary Figures and Tables Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7063434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497298720,"identity":"2840a037-5781-401b-93ca-d635a24ea5fc","order_by":0,"name":"Ralf 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We tested ADP / ADP-ribose analogs for inhibition of Mac1 of SARS-CoV-2 and determined affinity and binding mode of active compunds. The SAR showed, that replacement of either the distal ribose of ADPR by a small alkyl group or the adenine N7 by carbon improved inhibitory potency, resulting in the development of β-methyl-GS-441524-diphosphate, a nanomolar inhibitor that exhibits high selectivity (\u003e1000-fold) over human MacroD1 and MacroD2. Addition of C\u003csub\u003e11\u003c/sub\u003e-acyloxybenzyl (AB)-masking groups yielded a membrane permeable, lipophilic prodrug that inhibits SARS-CoV-2 in cell culture (EC\u003csub\u003e50\u003c/sub\u003e 0.06 μM) while exhibiting low cytotoxicity (CC\u003csub\u003e50\u003c/sub\u003e \u003e 50 μM). Replacement of the terminal methyl phosphate with an ethyl phosphonate increased stability of the prodrug with little effect on toxicity and antiviral potency (EC\u003csub\u003e50\u003c/sub\u003e = 0.03 μM), making it the first membrane-permeable nucleotide-based prodrug against viral macrodomains.","manuscriptTitle":"Targeting a viral macrodomain: Design, Structure-Based Optimization and Antiviral Evaluation of Nanomolar Inhibitors for Mac1 of SARS-CoV-2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 07:48:50","doi":"10.21203/rs.3.rs-7063434/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2fbcce4d-6937-4d0c-b58e-1fe85cd6c34c","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52829030,"name":"Biological sciences/Biochemistry/Enzymes"},{"id":52829031,"name":"Biological sciences/Drug discovery"},{"id":52829032,"name":"Biological sciences/Microbiology/Virology/SARS-CoV-2"}],"tags":[],"updatedAt":"2026-03-06T14:50:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 07:48:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7063434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7063434","identity":"rs-7063434","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}